Repetitive low dose thioacetamide (TA) treatment of hepatocytes was found to induce cells in G2 arrest. In the present study, an attempt was made to investigate alterations in expression of cell cycle regulators after G1 progression in the same repetitive low dose TA treated hepatocytes system and to define the determinators involved in G2 arrest. TA was daily administered intraperitoneally, with a dose of 50 mg/kg for 7 days. Expression levels of cyclin E and CDK2 were similar, increased at day 1 and reached a peak at day 2. And they recycled from day 3 reaching a second peak at day 5. Expression level of cyclin A was similar to p27(Kip1) and p57(Kip2) but not to CDK2 and increased to a peak level at day 2. Expression levels of cyclin B1 and cdc2 were similar although the cyclin B1 level was generally low, decreased from day 1 to basal levels at day 3 and persisted at a low level till day 7. The expression level of cyclin G1 was similar to p53 that peaked at day 3 and again at day 6 elevated over basal level. BrdU-labeled hepatocytic nuclei increased from 12 h, reached a peak at day 2, then decreased, and were not detectable from day 6. The number of PCNA-labeled nuclei increased immediately, peaked at day 2, and maintained till day 7. These results suggest that G2 arrest induced by repeated TA treatment might be p53-dependent, via activation of cyclin G1, rather than inhibition of cyclin B1- cdc2 complex, and inhibitors holding S phase progression might be p27(Kip1) and p57(Kip2).
Animals ; Bromodeoxyuridine/metabolism ; CDC2 Protein Kinase/drug effects/metabolism ; *CDC2-CDC28 Kinases ; Cell Cycle/drug effects/*physiology ; Cell Cycle Proteins/drug effects/metabolism ; Cyclin-Dependent Kinases/antagonists & inhibitors/drug effects/metabolism ; Cyclins/drug effects/metabolism ; Dose-Response Relationship, Drug ; G1 Phase/drug effects/*physiology ; Liver/*drug effects/pathology ; Male ; Nuclear Proteins/drug effects/metabolism ; Proliferating Cell Nuclear Antigen/metabolism ; Protein p53/metabolism ; Protein-Serine-Threonine Kinases/antagonists & inhibitors/drug effects/metabolism ; Rats ; Rats, Sprague-Dawley ; Thioacetamide/administration & dosage/*pharmacology ; Tumor Suppressor Proteins/drug effects/metabolism

Sphingosine kinase 1 (SphK1) plays critical roles in cell biological functions. Here we investigated the effects of SphK1 inhibitor SKI II on hepatoma HepG2 cell cycle progression and invasion. Cell survival was determined by SRB assay, cell cycle progression was assayed by flow cytometry, the ability of cell invasion was measured by Matrigel-Transwell assay and protein expression was detected by Western blotting. The results showed that SKI II markedly inhibited HepG2 cell survival in a dose-dependent manner, induced G1 phase arrest in HepG2 cell and inhibited cell invasion. SKI II markedly decreased the expressions of G1-phase-related proteins CDK2, CDK4 and Cdc2 and the levels of cell invasion-associated proteins MMP2 and MMP9. The results showed that SKI II inhibited cell cycle progression and cell invasion, implying SphK1 as a potential target for hepatoma treatment.
CDC2 Protein Kinase ; Cell Movement ; drug effects ; Cell Survival ; drug effects ; Cyclin-Dependent Kinase 2 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinases ; metabolism ; G1 Phase ; drug effects ; Hep G2 Cells ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; antagonists & inhibitors ; Thiazoles ; pharmacology

In the present study, a newly synthesized benzofuran lignan 4-formyl-2-(4-hydroxy-3methoxyphenyl)-5-(2-methoxycarbonyethyl)-7-methoxy-benzo [b] furan (ERJT-12) was tested for its antiproliferative activity on human tumor cells. The related mechanisms were also investigated. In vitro growth inhibitory effects of ERJT-12 on various cancer cell lines were determined by MTT assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The integrity of DNA was assessed by agarose gel electrophoresis. Activation of Caspase-3/7 and Caspase-6 was measured by colorimetric assay. The expressions of cell cycle proteins cell divide cycle 25c (Cdc25c), cyclin dependent kinase 1 (CDK1), CyclinB1 and apoptosis-related proteins Bax and Bcl-2 were detected by Western blotting. MTT assay showed that ERJT-12 inhibited the proliferation of several cancer cell lines including multidrug resistant cells. MCF-7 cells were markedly arrested at gap2/mitosis (G2/M) phase after treatment with ERJT-12 and progressed into apoptosis. The increased activities of Caspase-3/7 and Caspase-6 in MCF-7 cells were observed. The expression of CyclinB1 was down-regulated. The activities of Cdc25c and CDK1 protein were suppressed and Bcl-2 protein was phosphorylated. ERJT-12 displays potent antiproliferative activity towards cancer cells through suppressing cell cycle proteins, arresting cell cycle at G2/M phase and inducing apoptosis. It might be a novel candidate for cancer therapy.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Benzofurans ; pharmacology ; CDC2 Protein Kinase ; metabolism ; Caspase 3 ; metabolism ; Caspase 6 ; metabolism ; Caspase 7 ; metabolism ; Cell Cycle Proteins ; metabolism ; Cell Division ; drug effects ; Cell Line, Tumor ; Cyclin B ; metabolism ; Cyclin B1 ; G2 Phase ; drug effects ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism ; cdc25 Phosphatases ; metabolism

OBJECTIVETo elucidate the cytotoxicity and mechanism of 23-O-acetylcimigenol-3-O-beta-D-xylopyranoside isolated from C. dahurica on HepG2 cells and to find the leading compound for new drug development.

METHODMTT, AO/EB staining observation, flow cytometry and western blot methods were used to study the cytotoxicity, morphological changes, cell cycle distribution and protein expression profile of 23-O-acetylcimigenol-3-O-beta-D-xylopyranoside on HepG2 cells.

RESULT23-O-acetylcimigenol-3-O-beta-D-xylopyranoside could inhibit the proliferation of HepG2 cells with IC50 at 16 micromol x L(-1), and could also induce apoptosis and G2-M cell cycle arrest. Further study demonstrated that the compound could cleavage PARP, regulate protein expression of bcl-2 family and decrease the expression of cdc 2 and cyclin B.

CONCLUSION23-O-acetylcimigenol-3-O-beta-D-xylopyranoside exerts its cytotoxicity on HepG2 cells via apoptosis and G2-M arrest. In addition, caspases family activation, regulation of protein expression of bcl-2 family and down regulation of cdc 2 and cyclin B were involved in apoptosis and G2-M arrest induced by it.

Apoptosis ; drug effects ; CDC2 Protein Kinase ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cimicifuga ; chemistry ; Cyclin B ; metabolism ; Glycosides ; isolation & purification ; pharmacology ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Plants, Medicinal ; chemistry ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Triterpenes ; isolation & purification ; pharmacology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVEOur previous cDNA array data have shown that expression level of CDK1 increased along with the malignant progression of ganglioglioma, and decreased with the differentiation process of neural stem cells. The purpose of this study was to investigate the CDK1 expression levels in gliomas and the effects of CDK1 knockdown on phenotype of glioma cells.

METHODSGlioma tissue array was constructed, which was composed of surgical specimens of gliomas with different malignancy grades, glioma xenografts in nude mice, cellular spheroids of brain tumor stem cells, normal neural stem cells and glioma cell line. CDK1 expression was detected in glioma tissue array with immunohistochemical techniques. CDK1 expression in human brain glioma cell line and relevant xenogeneic graft tumor was inhibited by retroviral vectors expressing short hairpin RNAs (shRNAs). Both in vitro and in vivo changes of biological characteristics were further observed.

RESULTSThe expression level of CDK1 increased along with the malignancy progression of glioma in clinical specimens. The positive expression rates of CDK1 in human brain glioma tissues were 22.2% (grade I), 40.0% (grade II), 69.6% (grade III) and 78.6% (grade IV), P = 0.01, respectively. The positive expression rate of CDK1 in glioma cell line and implanted xenografts was similar as the clinical tumors with high malignancy, and higher than those in neural stem cells and brain tumor stem cells (P = 0.0014). Expression of CDK1 was high in human fetal brain tissues and bone marrows of nude mice, but low in normal adult human brain tissues. Downregulation of CDK1 inhibited the proliferation activities notably both in SHG-44 cells in vitro and relevant xenogeneic graft tumors, and induced apoptosis of tumor cells prominantly as well.

CONCLUSIONOverexpression of CDK1 may promote oncogenesis and progression of human gliomas. Downregulation of CDK1 expression can inhibit the proliferation activities of human malignant gliomas.

Animals ; Apoptosis ; drug effects ; Astrocytoma ; genetics ; metabolism ; pathology ; Brain Neoplasms ; genetics ; metabolism ; pathology ; Brain Stem Neoplasms ; metabolism ; CDC2 Protein Kinase ; genetics ; metabolism ; Cell Cycle ; drug effects ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Ganglioglioma ; genetics ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Glioma ; genetics ; metabolism ; pathology ; Humans ; Mice ; Mice, Nude ; Neoplasm Staging ; Neoplasm Transplantation ; RNA, Messenger ; metabolism

AIMTo determine the anti-angiogenic activity of emodin.

METHODSChick embryo assay and cultured endothelial cells were used.

RESULTSEmodin at doses of 150 and 300 microg/egg caused 37.6% and 63.2% inhibition of angiogenesis, respectively. Emodin was shown to inhibit the proliferation of primary cultured bovine aortic endothelial cells in the absence or presence of basic-fibroblast growth factor (bFGF) or the presence of vascular endothelial growth factor (VEGF) in a dose-dependent manner. The IC50 values by MTT assay were 5.56, 8.40 or 6.91 mg x L(-1), respectively. Emodin at concentrations from 5.4 to 21.6 mg x L(-1) induced apoptosis of endothelial cells for 37.6% to 72.6%. Emodin caused endothelial cell cycle arrest at G2/M phase. After emodin treatment, there was a down-regulation of Cyclin B1, P34cdc2, and Bcl-2 protein expression while the Bax protein expression was unaffected.

CONCLUSIONEmodin shows anti-angiogenic activity and might be useful for the development of novel anti-cancer therapy.

Animals ; Aorta ; cytology ; Apoptosis ; drug effects ; CDC2 Protein Kinase ; metabolism ; Cattle ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Cells, Cultured ; Chick Embryo ; Cyclin B ; metabolism ; Cyclin B1 ; Emodin ; pharmacology ; Endothelial Cells ; cytology ; metabolism ; Fibroblast Growth Factor 2 ; pharmacology ; Neovascularization, Physiologic ; drug effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Vascular Endothelial Growth Factor A ; pharmacology

Acute Lung Injury ; chemically induced ; metabolism ; pathology ; Animals ; CDC2 Protein Kinase ; genetics ; metabolism ; Cell Cycle ; drug effects ; Checkpoint Kinase 1 ; Female ; Lung ; metabolism ; pathology ; ultrastructure ; Male ; Malondialdehyde ; metabolism ; Microscopy, Electron, Transmission ; Organometallic Compounds ; toxicity ; Oxidative Stress ; Protein Kinases ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley

Silencing ATM gene gave rise to enhanced apoptotic response to irradiation and irradiation-like chemotherapy agents, this paper explored the crucial identities of the molecular elements responsible for the enhanced apoptotic response in U937 cells mediated by silencing ATM gene. Two U937 cell mutants named U937-ASPI3K (ATM, negative) and U937-pZeosv2(+) (ATM, wild-type) were used as a cell model system to identify the critical molecule(s) responsible for the varied apoptotic response in the absence or presence of ATM gene. Apoptosis was examined by measuring concentrations of free nucleosome in U937 cells. Western blot was employed to measure nuclear protein abundance of CDC25A, CDC25B, CDC25C, total p34cdc2, p34cdc2, (Thr 161) or p34cdc2 (Thr 14, Tyr 15). RT-PCR was used to estimate CDC25 transcript levels. U937-ASPI3K exhibited an enhanced apoptotic response to lower dosage of irradiation, which could not be blocked by protein synthesis inhibitor. Protein serine-threonine phosphatase inhibitor or cyclin-dependent kinase (CDK) inhibitors, on the other hand, abolished the enhancement indicated that protein phosphorylation/dephosphorylation modification and CDK activity are required for the enhanced apoptotic response in the absence of ATM gene. Upon irradiation, p34cdc2 in U937-pZeosv2(+) was maintained in an inactive state by phosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with a dramatic decrease of nuclear CDC25A, CDC25B and CDC25C proteins. In contrast, p34cdc2 in U937-ASPI3K maintained in an active state by dephosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with constant nuclear CDC25A, CDC25B and CDC25C protein abundance before and after irradiation. The responsive decrease of nuclear CDC25 proteins occurred at the post-transcription level. Silencing ATM gene blocks the responsive decrease of nuclear CDC25 proteins, which is responsible for failure to inactivate p34cdc2 after irradiation. Active p34cdc2 and CDK2, in turn, acts as the death executors to trigger apoptosis. In summary, aberrantly activated CDK activity is the critical molecular mechanism central to enhanced apoptotic responses in the absence of ATM gene.
Apoptosis ; drug effects ; genetics ; radiation effects ; Ataxia Telangiectasia Mutated Proteins ; CDC2 Protein Kinase ; metabolism ; Cell Cycle Proteins ; Cyclin-Dependent Kinases ; metabolism ; DNA-Binding Proteins ; Gene Silencing ; drug effects ; radiation effects ; Humans ; Protein-Serine-Threonine Kinases ; genetics ; Tumor Suppressor Proteins ; U937 Cells ; cdc25 Phosphatases ; metabolism

OBJECTIVETo explore the molecular mechanisms of G(2)/M checkpoint initiated by diallyl disulfide (DADS) in HL-60 cells.

METHODSCell viability was determined by MTT assay. Cell cycle was assayed by flow cytometry. The expression of phospho-p38, Cdc25B and Cdc2, and p38 mRNA were measured by Western blotting and RT-PCR, respectively.

RESULTSAfter treatment with DADS at 5 - 160 micro mol/L for 0 - 72 h, the growth of HL-60 cells were suppressed in a concentration-dependent manner and the inhibitory effect of DADS (20 micro mol/L) was similar to that of ATRA (10 nmol/L) (P > 0.05). Incubation of HL-60 cells with DADS (20 micro mol/L) for 12 h could activate G(2)/M checkpoint and increase the expression of phospho-p38 MAPK, followed by the expression of phospho-Cdc25B and phospho-Cdc2 (P < 0.05). SB202190, a specific inhibitor of p38 MAPK, markedly blocked the phosphorylation of p38 MAPK, Cdc25B and Cdc2 (P < 0.05).

CONCLUSIONDADS could induce the G(2)/M arrest in HL-60 cells which may be involved in the activation of p38 MAP kinase.

Allyl Compounds ; pharmacology ; Blotting, Western ; CDC2 Protein Kinase ; genetics ; metabolism ; Cell Division ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Disulfides ; pharmacology ; Dose-Response Relationship, Drug ; Enzyme Activation ; drug effects ; Flow Cytometry ; G2 Phase ; drug effects ; Gene Expression ; drug effects ; HL-60 Cells ; Humans ; Phosphorylation ; drug effects ; Reverse Transcriptase Polymerase Chain Reaction ; cdc25 Phosphatases ; genetics ; metabolism ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

AIMTo study the antitumor mechanism of 3-substituted aryl oxindole (PH II-7) and determine its effects on cell cycle distribution of tumor cells.

METHODSThe cell cycle distributions were determined with FACS. The cell cycle regulation-related proteins of K562 lysates were analyzed with Western Blot. The inhibition of PH II-7 on DNA synthesis of tumor cells were estimated though 3H-thymidine incorporation and the tyrosine kinase activity of EGFR of A431 lysates was measured with ELISA.

RESULTSPH II-7 effected cell cycle distribution of several tumor cells, including multidrug resistant tumor cell lines, and accumulation of cells in the G0-G1 stages was observed. The cell cycle regulation-related proteins CDK2, Rb and c-myc were inhibited by PH II-7 in a dose dependent manner, whereas the expression of CyclinE was increased after exposure to PH II-7. Furthermore, PH II-7 2.0 mg.L-1 was shown to inhibit the incorporation of 3H-thymidine into DNA, and 21.89%-41.29% of the PTK activity of EGFR in A431 lysates was inhibited by PH II-7 2-8 mg.L-1 in a dose-dependant manner.

CONCLUSIONPH II-7, a new anti-tumor agent, blocks the transition of cell cycle of tumor cells from G1 to S phase by inhibition CDK2.

Antineoplastic Agents ; pharmacology ; CDC2-CDC28 Kinases ; metabolism ; Cell Cycle ; drug effects ; Cell Cycle Proteins ; metabolism ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase 2 ; DNA, Neoplasm ; biosynthesis ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Humans ; Indoles ; chemical synthesis ; pharmacology ; K562 Cells ; pathology ; Proto-Oncogene Proteins c-myc ; metabolism ; Retinoblastoma Protein ; metabolism